2-Amino-3-benzylthiophenes such as PD81723 (PD) have been shown to be selective allosteric enhancers of A1 adenosine receptors. At high concentrations these compounds act as competitive antagonists. Our preliminary data indicate that PD enhances the coupling of recombinant human A1 receptors to G proteins, but PD has no enhancing effect on other human adenosine receptors. A1 enhancers have great therapeutic potential since they may produce minimal side effects or desensitization and act selectively in ischemic tissues at sites where endogenous adenosine is produced. Of six aims, four are directed at determining the molecular mechanisms by which PD causes allosteric enhancement of human A1 adenosine receptors. We have stably expressed all four human adenosine receptor subtypes, and modified these receptors by extending the amino termini with hexahistidine and the FLAG epitope to make H/F- receptors that can readily be purified. PD enhances agonist binding to purified H/F-A1 receptors, proving that it binds directly to an allosteric site on the receptor. We have prepared adenosine receptor chimera and mutants, several unique radioligands and photoaffinity labels, and homogeneous recombinant G proteins. Aim 1 is to evaluate the enhancing and antagonist activities of PD on all four recombinant human adenosine receptors. Aim 2 is to determine the effects of PD on the interaction between purified H/F-A1 receptors and purified G proteins of defined subunit composition in reconstitution assays. Aim 3 is to examine the effects of PD on an A1/A2A hybrid receptor we have constructed that displays A1 pharmacology and Gs coupling (A1 and A2A receptors coupled to Gi and Gs, respectively). Aim 4 is to construct A1/A3 receptor chimera and A1 mutants to identify amino acids required for enhancer activity. Two additional aims will be facilitated by the participation of the co-investigator, Dr. Olsson, an expert medicinal chemist. Aim 5 is to synthesize and screen new compounds that are more potent and possibly more efficacious enhancers than PD based on binding, biochemical and functional (Langendorff perfused guinea pig heart) assays. It may be possible to synthesize enhancers with little or no antagonist activity. Aim 6 is to synthesize enhancer radioligands and photoaffinity labels for use in characterizing the enhancer binding domain. Affinity labeled purified receptors will be sequenced to identify labeled amino acids. In addition to contributing to the possible development of promising therapeutic agents, these studies will be useful for increasing our understanding of factors that regulate receptor-G protein coupling.